NO321866B1 - Process for preparing a phospholipid suspension. - Google Patents

Abstract

The present invention describes processes for the preparation of a lipid composition and a homogeneous filterable phospholipid suspension containing the lipid composition, wherein such a suspension is useful as an ultrasonic contrast agent.

Description

The present invention relates to a method for producing a homogeneous filterable phospholipid suspension containing a lipid mixture, whereby a suspension is obtained which is useful as an ultrasound contrast agent.

Preparation of a phospholipid contrast agent can be divided into the following steps: (1) preparation of lipid mixture; (2) mixing the bulk solution, which involves hydration and dispersion of the lipid mixture in a largely aqueous medium to produce a lipid suspension; (3) filtering the bulk solution through a sterilizing filter(s) to render the suspension free of microbial contaminants; (4) dispense the sterile suspension into individual vials in a controlled aseptic area; (5) loading the assigned vials into a lyophilization chamber to replace the gas at the top of the vial with perfluoropropane (PFP) gas; (6) transferring the closed medicinal vials after gas exchange to an autoclave for final sterilization. There are three main obstacles to this method: (1) homogeneity of the lipid mixture; (2) the hydration of the lipid mixture; (3) homogeneity and particle size of the suspension; and (4) sterile filtering the suspension through a sterilizing filter(s).

Phospholipid mixtures are typically prepared by dissolving or suspending the required lipids in a suitable aqueous or non-aqueous solvent system, and then reducing the volume either by lyophilization or distillation. Ideally, this method produces mixed solids of high homogeneity and purity. However, while it works well on a small, laboratory scale, this simple approach is often problematic when scaling up to production-size quantities. Difficulties include: (1) maintaining homogeneity throughout the solvent removal step (due to different solubilities); (2) maintaining purity (often a problem when water is used due to hydrolytic side reactions); (3) to improve purity; (4) to minimize solvent volume; and (5) recovery of the final solids (eg, it is not practical to scrape out solids from a large reactor).

After preparation of a lipid mixture, final mixing typically involves introducing the mixture into an aqueous medium. Since phospholipids are hydrophobic and are not readily soluble in water, adding phospholipids or a lipid mixture directly to a solution causes the lipid powder to aggregate and form clumps that are very difficult to disperse. Thus, the hydration process cannot be controlled within a reasonable operating period. Direct hydration of phospholipids or a lipid mixture in an aqueous medium yields a cloudy suspension with particles ranging from 0.6 (im to 100 µm. Because of the relatively large particle size distribution, the suspension cannot be filtered at room temperature when the suspension solution temperature is below the gel-to - the liquid crystal phase transition temperatures for lipids. The lipids would accumulate in the filters which would cause a limitation in the flow rate, and in most cases the filters would be completely blocked soon after. Further reduction in the suspension particle size cannot be achieved through a conventional mixing process ("batching process "), even after prolonged mixing (eg 6 hours) at elevated temperatures (eg 40°C to 80°C) with a commonly used marine propeller.

Although filtration at elevated temperatures, i.e. at temperatures above the phase transition temperatures for lipids, is possible, a significant amount of large lipid particles would still be excluded when a normal filtration pressure is used. Furthermore, concentrations of the sterile filtrate would have variable lipid contents from batch to batch depending on how the lipids are initially hydrated, which in turn is determined by the physical characteristics, for example morphology, of the starting materials.

The process of directly hydrating the lipids or lipid mixture to produce a homogeneous suspension and filtering the suspension through a sterilizing filter(s) can be difficult and expensive to scale up to a reasonable commercial scale, eg >201.

Thus, the present method for producing a phospholipid suspension, which method includes the step of producing a lipid mixture, aims to solve the problems discussed above by providing a practical method that can be easily scaled up and adapted to different production facilities without expensive modification or individual adaptation of existing equipment.

Accordingly, an aim of the present invention is to provide a new method for producing a phospholipid suspension including a prior step of producing said lipid mixture.

This and other objects which will become apparent through the following detailed description have been achieved by the inventors' discovery that dissolving a lipid mixture in a suitable non-aqueous solvent prior to the introduction of an aqueous solution allows for the production of a phospholipid suspension.

[1] Thus, the present invention provides a method for producing a phospholipid suspension, and this method is characterized by the fact that it comprises:

(a) contacting at least two lipids with a first non-aqueous solvent to form a solution; (b) concentrating the solution to a thick gel; (c) contacting the thick gel with a second non-aqueous solvent to form a solution; (d) concentrating this solution from step (c) to form a lipid mixture; (e) contacting the lipid mixture with a non-aqueous solvent, wherein the lipid mixture is substantially dissolved in the non-aqueous solvent to form a

salary; and

(f) without removing the non-aqueous solvent, contacting the solution from step (e)

with an aqueous solution to form a lipid suspension.

[2] In a preferred embodiment, the non-aqueous solvent is selected from propylene glycol, ethylene glycol and polyethylene glycol 300.

[3] In a more preferred embodiment, the non-aqueous solvent is polypropylene glycol.

[4] In another embodiment, the lipid mixture comprises: (a) 1,2-dipalnutoyl-,s/z-glycero-3-phosphatidylcholine; (b) 1,2-dipalmitoyl-s'-glycero-3-phosphotidine, mono sodium salt; and (c) N-(methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-jw-glycero-3-phosphatidylethanolamine, mono sodium salt.

[5] In another preferred embodiment, the non-aqueous solvent in step (1) is heated to a temperature of about 30 to 70°C before being contacted with the lipid mixture.

[6] In another preferred embodiment, the non-aqueous solvent is heated to a temperature of about 50 to 55°C before being contacted with the lipid mixture.

[7] In another preferred embodiment, the ratio of the lipid mixture to the non-aqueous solvent is from about 5 mg of the lipid mixture per ml of non-aqueous solvent to about 15 mg/ml.

[8] In another preferred embodiment, the ratio of lipid mixture to non-aqueous solvent is about 10 mg/ml.

[9] In another preferred embodiment, the aqueous solution in step (2) is selected from water, salt solution, a salt solution/glycerin mixture and a salt solution/glycerin/non-aqueous solvent mixture.

[10] In a more preferred embodiment, the aqueous solution is a salt solution and glycerin mixture.

[11] In another preferred embodiment, the aqueous solution is a salt solution, glycerin and propylene glycol mixture.

[12] In another more preferred embodiment, 6.8 mg/ml of sodium chloride is present, 0.1 ml/ml of glycerin is present, 0.1 ml/ml of propylene glycol is present, and about 0.75 to 1.0 mg /ml of the lipid mixture present.

[13] In an even more preferred embodiment, 0.75 mg/ml of the lipid mixture is present.

[14] In another more preferred embodiment, 1.0 mg/ml of the lipid mixture is present.

[15] In another preferred embodiment, the aqueous solution in step (f) is heated to a temperature of about 45 to 60°C before being contacted with the solution from step (e).

[16] In another more preferred embodiment, the aqueous solution is heated to a temperature of about 50 to 55°C before contacting it with the solution from step (e).

[17] In another preferred embodiment, the method further comprises: (g) heating the lipid suspension from step (f) to a temperature approximately equal to or higher than the highest gel-to-liquid crystal phase transition temperature of the lipids present in the suspension.

[18] In another more preferred embodiment, the lipid suspension is heated in step (g) to a temperature of at least about 67°C.

[19] In another, more preferred embodiment, the method further comprises:

(h) filtering the lipid suspension through a sterilizing filter.

[20] In another even more preferred embodiment, the filtration is carried out in step (h) by using two sterilization filter inserts.

[21] In a further preferred embodiment, the sterilization filter inserts in step (h) are at a temperature of from approximately 70 to 80°C.

[22] In another further preferred embodiment, 0.2 nm hydrophilic filters are used in step (h).

[23] In another even more preferred embodiment, the method further comprises:

(i) dispensing the filtered solution from step (h) into a vial.

[24] In another further preferred embodiment, the method further comprises: (j) exchanging the gas in the upper part of the medicine glass from step (i) with a perfluorocarbon gas.

[25] In another even further preferred embodiment, the perfluorocarbon gas is perfluoropropane.

[26] In another even further preferred embodiment, the replacement of the upper gas is carried out by using a lyophilization chamber.

[27] In another even further preferred embodiment, the method further comprises:

(k) sterilizing the vial from step (j).

[28] In a still further preferred embodiment, the vial is sterilized in step (k) at approximately 126-130°C for 1 to 10 minutes.

[29] In another preferred embodiment, the first non-aqueous solvent in step (a) is a mixture of methanol and toluene.

[30] In another preferred embodiment, the second non-aqueous solvent in step (c) is methyl t-butyl ether.

[31] In another preferred embodiment, the solution in step (a) is heated to a temperature sufficient to complete the dissolution of the lipids in the solvent.

[32] In another more preferred embodiment, the solution in step (a) is heated to about 25 to 75°C.

[33] In another preferred embodiment, the solids collected in step (d) are washed with methyl t-butyl ether and dried under vacuum.

The phospholipid suspension obtained by the present method may conveniently comprise: (a) a lipid mixture in an amount of approximately 0.75-1.0 mg/ml of suspension; (b) sodium chloride in an amount of about 6.8 mg/ml of suspension; (c) glycerin in an amount of about 0.1 ml/ml of suspension; (d) propylene glycol in an amount of about 0.1 ml/ml of suspension; and (e) water, and this suspension can be prepared by: (1) contacting a lipid mixture with a non-aqueous solvent, wherein the lipid mixture is substantially dissolved in the non-aqueous solvent; (2) contacting the solution from step (1) with an aqueous solution to form a lipid suspension; (3) heating the lipid suspension from step (2) to a temperature approximately equal to

or higher than the highest gel-to-liquid crystal phase transition temperature of the lipids present in the suspension; and

(4) filtering the lipid suspension through a sterilizing filter.

Formulation

The present invention is intended to be performed on at least a multigram scale, kilogram scale, multikilogram scale, or industrial scale. Multigram scale, as used here, is preferably the scale where at least one starting material is present in 10 g or more, more preferably at least 50 g or more, even more preferably at least 100 g or more. Multi-kilogram scale, as used here, is intended to mean the scale where more than 1 kg of at least one starting material is used. Industrial scale, as used herein, is intended to mean a scale other than laboratory scale that is sufficient to supply product sufficient for either clinical testing or distribution to consumers. Lipid mixture or phospholipid mixture as used here is meant to represent two or more lipids that have been mixed. The lipid mixture is generally in a powder form. Preferably, at least one of the lipids is a phospholipid. Preferably, the lipid mixture contains 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-j/i-glycero-3-phosphotidine, mono sodium salt (DPPA), and N-(methoxypolyethylene glycol 5000 carba -moyl)-1,2-dipalmitoyl-j/i-glycero-3-phosphatidylethanolamine, mono sodium salt (MPEG5000-DPPE). The amount of each lipid present in the mixture will depend on the desired end product. Preferred ratios of each lipid are described in the examples section. A wide variety of other lipids, such as those described in Unger et al., U.S. patent no. 5,469,854, the content of which is incorporated here as a reference, can be used in the present method.

Phospholipid, as used here, is a fatty substance containing an oily (hydrophobic) hydrocarbon chain(s) with a polar (hydrophilic) phosphoric acid main group. Phospholipids are amphiphilic. They spontaneously form boundaries and close voids in aqueous media. Phospholipids comprise about 50% of the amount of animal cell plasma membrane.

Preparation of the lipid mixture

The lipid mixture can be prepared via an aqueous suspension-lyophilization method or an organic solvent dissolution-precipitation method using organic solvents. In the aqueous suspension-lyophilization method, the desired lipids are suspended in water at an elevated temperature and then concentrated by lyophilization. A dissolution method is preferably used.

Step (a):

The organic solvent dissolution-precipitation method involves contacting the desired lipids (eg, DPPA, DPPC, and MPEG5000 DPPE) with a first non-aqueous solvent system. This system is typically a combination of solvents, for example CHCl3/MeOH, CH2Cl2/MeOH and toluene/MeOH. Preferably, the first non-aqueous solvent is a mixture of toluene and methanol. It may be desirable to heat the lipid solution to a temperature sufficient to achieve complete dissolution. Such a temperature is preferably about 25 to 75°C, more preferably about 35 to 65°C.

After dissolution, it may be desirable to remove undissolved foreign material by hot filtration or cooling to room temperature and then filtration. Known methods of filtration can be used (for example gravity filtration, vacuum filtration or pressure filtration).

Step (b):

The solution is then concentrated to a thick gel/semi-solid. The concentration is preferably carried out by vacuum distillation. Other methods for concentrating the solution, such as rotary evaporation, can also be used. The temperature in this step is preferably about 20 to 60°C, more preferably 30 to 50°C.

Step (c):

The thick gel/semi-solid is then dispersed in a second non-aqueous solvent. The mixture is slurried, preferably close to room temperature (for example 15-30°C). Other useful non-aqueous solvents are those which cause the lipids to precipitate from the filtered solution. The second non-aqueous solvent is preferably methyl t-butyl ether (MTBE). Other ethers and alcohols can also be used.

Step (d):

The solids produced by the addition of the second non-aqueous solvent are then collected. The collected solids are preferably washed with another portion of the second non-aqueous solvent (eg MTBE). Collection can be carried out via vacuum filtration or centrifugation, preferably at room temperature. After collection, it is preferred that the solids are dried under vacuum at a temperature of approximately 20 to 60°C.

For the following reasons, the organic solvent dissolution-precipitation method is preferred over the aqueous suspension/lyophilization method: (1) Because the lipids are quite soluble in toluene/methanol, the solvent volumes are greatly reduced (relative to the aqueous procedure). (2) Due to this increased solubility, the operating temperature is also lower relative to the aqueous procedure, thereby avoiding the hydrolytic instability of fatty acid esters. (3) When cooled back to room temperature, the toluene/methanol solution of lipids remains homogeneous, allowing a room temperature filtration to remove solid foreign material. (4) The MTBE precipitation provides rapid and easy isolation of lipid mixture solids. With the aqueous method, a time-consuming lyophilization method is used to isolate the material. (5) The MTBE precipitation also allows for the removal of any MTBE-soluble impurities, which are introduced into the filtrate waste stream. This possibility of impurity removal is not achieved when a solution is concentrated directly or lyophilized to a solid.

(6) The present process gives homogeneous solids.

Preparation of the lipid suspension

Step (é):

In step (e), a lipid mixture is contacted with a non-aqueous solvent, whereby the lipid mixture is essentially dissolved in the non-aqueous solvent. Alternatively, the individual lipids can be contacted with the non-aqueous solvent sequentially in the order: DPPC, DPPA and MPEG5000-DPPE; DPPC, MPEG5000-DPPE and DPPA; MPEG5000-DPPE, DPPA and DPPC; or MPEG5000-DPPE, DPPC and DPPa. DPPA, which is the least soluble and least overwhelming of the lipids, is not added first. Adding one of the other lipids before or at the same time as adding DPPA facilitates the dissolution of DPPA. In another alternative, the individual lipids can be combined in their solid forms and the combination of the solids contacted with the non-aqueous solvent.

Substantial dissolution is generally indicated when the mixture of lipid mixture and non-aqueous solvent becomes clear. As noted earlier, phospholipids are generally not water soluble. Thus, direct application of a mixture of phospholipid mixture in an aqueous environment causes the lipid mixture to aggregate to form clumps which are very difficult to disperse. The present invention overcomes this limitation by dissolving the lipid mixture in a non-aqueous solvent before the aqueous solution is added. This makes it possible to disperse the lipid mixture evenly in a liquid. The liquid dispersion can then be added to a desired aqueous environment.

Non-aqueous is intended to mean a solvent or mixture of solvents in which the amount of water present is sufficiently low not to impede dissolution of the lipid mixture. The amount of non-aqueous solvent required will depend on the solubility of the lipid mixture and also the final desired concentration of each component. As one skilled in the art will appreciate, the level of water present in the non-aqueous solvent that can be tolerated will vary based on the water solubilities of the individual lipids in the lipid mixture. The more water soluble the individual phospholipids are, the more water can be present in step (1). Propylene glycol is preferably used as the non-aqueous solvent. However, other members of the polyol family, such as ethylene glycol, and polyethylene glycol 300 can be used.

Mechanical mixing of the lipid mixture and the non-aqueous solvent may be necessary to achieve complete dissolution. One skilled in the art will appreciate that a variety of mixing methods are available. It is preferred that a powerful cutting homogenisation apparatus is used.

One skilled in the art will appreciate that increasing the temperature of the solvent will aid in dissolving the lipid mixture. The temperature at which step (1) can be carried out can range from room temperature to the boiling point of the chosen solvent. Preferably, the temperature is from about 30 to about 70°C, more preferably about 45 to about 60°C, and even more preferably about 50, 51, 52, 53, 54 or 55°C. When ethylene glycol or polyethylene glycol 300 is used, it is preferred that the temperature is from about 50 to about 60°C and more preferably about 55°C. By keeping the solution at an elevated temperature, solution viscosity will be reduced and formulation preparation will be simplified.

A preferred method for dissolving the lipid mixture is as follows: (i) Add propylene glycol to a suitable weighing container. (ii) Heat the polypropylene glycol to about 40 to 80°C in a heat bath. (iii) Weigh the lipid mixture into a separate container, (iv) When the polypropylene glycol has reached the desired temperature range, transfer the solution to the container containing the lipid mixture. (v) Place the container back into the heat bath until the solution becomes clear. (vi) Mechanically mix the lipid mixture/propylene glycol solution to further ensure complete dissolution and homogeneous dispersion of the lipid mixture.

The ratio of lipid mixture to non-aqueous solvent will of course be limited by the solubility of the lipid mixture. This ratio will also be influenced by the desired amount of lipid mixture in the final formulation. Preferably, the ratio is from about 1 mg of lipid mixture per ml of solvent (mg/ml) to about 100 mg/ml. More preferably, the lipid mixture is present at about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mg/ml. Even more preferably, the lipid mixture is present at about 10 mg/ml.

Steps ff):

The second step involves contacting the solution from step (e) with an aqueous solution to form a lipid suspension. The aqueous solution can be water, saline, a saline/glycerin mixture, or a saline/glycerin/non-aqueous solvent mixture. Non-aqueous solvent is, as defined earlier, preferably propylene glycol. Suspension, as used here, is intended to indicate a dispersion in which insoluble particles are dispersed in a liquid medium.

Once complete dissolution of the lipid mixture is achieved (step (e)), that solution can then be added to an aqueous solution. The aqueous solution may contain one or more components selected from sodium chloride, glycerin, and a non-aqueous solvent. Preferably, the aqueous solution contains glycerin and sodium chloride. Preferably, a sufficient amount of propylene glycol is present in the aqueous solution before the addition of the solution from step 1, to achieve the final desired concentration of propylene glycol.

The order of addition of desired components is not expected to seriously affect the lipid suspension obtained. However, it is preferred that the lipid mixture solution is added to water, which may already contain the above-noted additional components. Additional desired components can then be added. It is more preferred that the lipid mixture solution is added to a solution of water and sodium chloride (ie saline). It is further preferred that the lipid mixture solution is added to a solution of water, sodium chloride and glycerin. It is even more preferred that the lipid mixture solution is added to a solution of water, sodium chloride, glycerin and propylene glycol.

It is preferred by 6.8 mg NaCl is present per ml formulation. Preferred is 0.1 ml of glycerin per ml formulation present. A final concentration of 0.1 ml propylene glycol per ml formulation is preferred. The final pH of the formulation is preferably about 5.5-7.0. The lipid mixture is preferably present in an amount of 0.75-1.0 mg/ml in relation to the formulation.

The temperature of the aqueous solution can range from room temperature to 70°C. The preferred temperature is about 45 to 60°C, with 50, 51, 52, 53, 54 or 55 being even more preferred. To achieve complete dissolution, the mixture will need movement, preferably stirring. The pH of the solution may also need to be adjusted, depending on the desired final formulation. Either acid (eg, HC1) or base (eg, NaOH) can be added to achieve such an adjustment.

The lipid suspension will contain liquid particles of varying sizes. One of the advantages of the present invention is the ability to consistently obtain small particles of almost homogeneous size. Thus, it is preferred that the majority of particles obtained are less than 100 nm in diameter, more preferably less than 50 nm.

A preferred method for dissolving the lipid mixture is as follows: (i) Add water for injection (WFI) to a mixture formulation container. (ii) Starting the mixture and ensuring that the temperature is from 50 to 55°C. (iii) Add sodium chloride to the mixture formulation container. Wait until the solid has completely dissolved before proceeding to the next step, (iv) Add glycerin to the compound formulation container. Allow sufficient time for complete mixing, (v) Add the remaining propylene glycol not in the lipid mixture/propylene glycol solution. Allow time for thorough mixing, (vi) Reduce mixing speed to reduce turbulence in the mixing formulation vessel. (vii) Add the lipid mixture/propylene glycol solution to the mixture formulation vessel. (viii) Return mixing to original speed (ix) Add additional WFI if necessary, (x) Continue mixing for approximately 25 minutes and ensure complete mixing, (xi) Check and adjust solution to specified pH.

Step (g):

Step (g) involves heating the lipid suspension obtained in step (f) to a temperature approximately equal to or above the highest gel-to-liquid crystal phase transition temperature of the lipids present in the solution.

One of the purposes of this step is to provide a filterable suspension. A solution/suspension is considered to be filterable if there is no perceptible reduction in the flow rate by normal procedure, and there is no perceptible increase in the pressure drop in the filtration system.

Experimental data indicate that the lipids in the formulation should be below their gel-to-liquid crystal phase transition to facilitate sterile filtration. When the lipids are below the phase transition temperature, the suspension particles are rigid. However, when they are above their respective gel-liquid crystal phase transition temperatures, they are in a more soluble organized configuration and thus easier to filter.

DPPC and DPPA show phase transitions of respectively 41°C and 67°C. MPEG5000-DPPE is soluble in water and therefore does not exhibit a gel-liquid crystal phase transition characteristic of most hydrated lipid suspensions. Because all the lipids in the preferred formulation exhibit different gel-to-liquid phase transitions, the highest phase transition temperature, 67°C, is preferably used to filter the solution. By maintaining the temperature at or below 67°C, all the lipids are under their respective phase transitions, ensuring soluble configuration as they pass through the filters.

Heating can be achieved by enclosing the mixture formulation container with a heat exchange coil. Hot water/steam from a controlled source, e.g. a hot water bath, or a water heater, would supply sufficient heat to maintain the compound formulation solution at a given temperature. Other heat sources known to those skilled in the art can also be used.

Step (fr):

Step (h) is carried out by filtering the liquid suspension through a sterilizing filter. The purpose behind this step is to provide a largely bacteria-free suspension. A filtrate is considered largely bacteria-free when the probability that the filtrate contains at least one colony-forming microorganism is less than 10"<6>.

Filtration is preferably carried out by using sterilization filter inserts. A means of forcing the solution through the filters may also be necessary (eg pumping or pressurizing). Since the solution being filtered needs to be maintained at a temperature at or above the highest gel-to-liquid crystal phase transition temperature of the lipids present in the solution, the filtration should be performed at approximately these same temperatures. To achieve this, the filter (e.g. the sterilizing filter inserts) is preferably enclosed in enclosed filter housings which are continuously heated, e.g. by a hot water stream from a temperature-controlled heating bath, to ensure that the suspension is above the lipid phase transition temperatures. The temperature of the sterilizing filter is preferably from 50 to 100°C, more preferably from 60 to 90°C, and even more preferably 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80°C.

One or more sterilization filters can be used to filter the suspension. The necessary number will be based on their effectiveness in removing bacteria. It is preferred that two filters are used. The size of the filter pores will be limited by the need to provide a bacteria-free suspension. 0.2 µm hydrophilic filters are preferably used.

A bulk solution of the preferred formulation was continuously filtered through two 0.2 µm hydrophilic filters for up to 3 hours at a rate of approximately 11 per minute (1 l/min.), i.e. passing a total of 1,801 of the suspension solution through the filters. The experimental results show that there is no visible blockage of the filters. Lipid studies show that there is no measurable loss during the filtration process (due to accumulation in the filter medium).

A bulk solution of the preferred formulation was mixed at 40-80°C and the suspension was cooled to room temperature before sterile filtration. No visible clogging of the filters was observed, indicating that the suspension particle size distribution is well below 0.2 µm of the filter pore size. It is desirable to use heat throughout the filtration to ensure maximum recovery of the lipid mixture in the sterile filtrate (ie to minimize potential retention of lipid particles in the filter medium).

A preferred method for filtering lipid suspensions is as follows: (a) Ensure all encapsulated filters are at 70-80°C. (b) Ensure that all valves in the filtration unit are closed, (c) Connect the filtration inlet hose to the outlet of the compound formulation vessel. (d) Open valves to allow solution to pass through the filters. (e) Flush three liters of solution through the filters before collecting the filtrate. (f) Continue filtering until complete.

Step ( i ) :

Transferring the filtered solution to a vial completes step (i). This step is preferably carried out in a controlled antiseptic area. A person skilled in the art will understand that the vial selected and the amount of suspension filled into the vial will depend on the intended use of the lipid suspension. The transfer can be achieved through a variety of methods, including pipette, hand-held syringe container (eg, Filamatic® Syringe Dispenser), or industrial auto-dispensing machine (eg, Cozzoli or TL auto-filler).

Step (i):

Step (j) is carried out by exchanging the gas at the top of the medicine glasses from step (i) with a perfluorocarbon gas. A preferred method of exchange is to load the allocated vials into a lyophilization chamber and exchange the upper gas in the vial with a perfluorocarbon gas. A preferred gas is perfluoropropane (PFP). Other methods for exchanging the upper gas known in the art can be used.

The medication vials are sealed upon completion of the gas exchange cycle. When the lyophilization chamber pressure has been brought back to atmospheric pressure by charging the chamber with PFP. Medicine vial stoppers are placed to seal the medicine vials.

Step (k):

Step (k) involves final sterilizing a vial after step (j). One method for final sterilization is through the use of an autoclave. Also the sealed medicine jars can be final sterilized in a steam sterilization unit to further improve the sterility safety of the product. Care must be taken in the sterilization process as degradation of lipids may be observed as a result of autoclaving. Preferably, the vial is sterilized at about 126-130°C for 1 to 10 minutes.

Other features according to the invention will be revealed by the following description of embodiments which are to serve as examples which are given to illustrate the invention and are not intended to be limiting of it.

EXAMPLES

A flask is charged with toluene (3.3 L), methanol (1.2 L), DPPA (59.6 g), DPPC (535 g), and MPEG5000 DPPE (405 g). After rinsing solid contact surfaces with 0.91 methanol, the slurry is heated to 45-55°C until dissolution is complete.

The solution is filtered and then concentrated under vacuum at 35-45°C to a thick gel. Methyl t-butyl ether (MTBE, 5.41) is added and the mixture is slurried at 15-30°C. White solid is collected by centrifugation or vacuum filtration, and washed with MTBE (0.9 L). The solids are then placed in a vacuum oven and dried to a constant weight at 40-50°C. The dried lipid mixture is transferred to a bottle and stored at -15 to -25°C.

In another embodiment of the lipid mixture production procedure according to the present invention, the following procedure can also be used.

Alternative lipid mixture production procedure

Phospholipid amounts were adjusted for purity based on an "as used" value from the certificates of analysis. The batch size (pooled phospholipid weight) for this experiment was 2 kg.

A rotary evaporation flask is charged sequentially with toluene (3,300 mL), methanol (1,200 mL), DPPA (122.9 g; corrected for "as used" purity of 97.0%), DPPC (1098.5 g total; 500.8 g from a batch with 98.4% "as-used" purity and 5597.7 g from a batch with 96.7% "as-used" purity), and MPEG5000 DPPE (815.7 g, corrected for "as-used" purity of 99.3%). After rinsing the remaining solid in the flask with methanol (900 mL), the flask is placed on a rotary evaporator (not vacuum) and the slurry is heated to between 45 and 55°C (externally). After dissolution is complete, the external temperature is lowered to between 35 and 45°C, and vacuum is applied, and the solution is concentrated to a white semi-solid. The flask is removed from the evaporator and solids are loosened with a spatula. The flask is placed back on the evaporator and concentration continues. After the end point is reached (final vacuum pressure 20 mbar; white, granular, thick solid), MTBE (5,400 mL) is added through the addition tube of the rotary evaporator, the vacuum is interrupted, and the mixture is slurried for 15 to 45 minutes at 15 to 30°C. Solid is isolated by either centrifugal or vacuum filtration, rinsed with MTBE (3800 mL), and dried to constant weight in a vacuum oven (40 to 50°C). Prior to transfer to polyethylene bottles with polypropylene caps, solids are passed through a screen (0.079 inch mesh) yielding 1966.7 g (98%) of lipid mixture (SG896) as a white solid.

The preferred lipid suspension contains:

1,2-dipalmitoyl-jw-glycero-3-phosphotidine, monosodium salt (DPPA); 1,2-dipalmitoyl-1β-glycero-3-phosphatidylcholine (DPPC);

N-(Methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-jn-glycero-3-phosphatidylethanolamine, monosodium salt (MPEG5000-DPPE);

Propylene Glycol, USP;

Glycerin, USP;

Sodium Chloride, USP; and

Water for injection, USP.

The finished product's filling volume can be from 1.0 to 2.0 ml/medication glass.

In the preparation of the preferred formulation, when the lipid mixture is directly hydrated with the aqueous matrix solution comprising water for injection, sodium chloride, glycerin and propylene glycol, the filtrates have less lipids compared to the bulk solution before filtration. The loss of lipids varies from 12% to 48%. These results show that the sterile filtration procedure is not effectively controlled and therefore the final product lipid content is highly variable.

In contrast, by using the present described method, experimental results of the lipids show complete recovery of lipids through the filtration method. The variability of experimental results around the theoretical targets is within normal experimental method variability. Particle size distribution based on number, based on volume and based on reflective intensity of a suspension prepared by first dissolving the lipid mixture in propylene glycol indicates that the majority of particles are smaller than 50 nm in the bulk solution before filtration at 55°C as well as at 70° C. The particle distribution profile does not change after filtration.

EXPLOITATION PART

The present method is useful for producing ultrasound contrast agents. Such agents should be useful for a variety of imaging applications, including enhancing contrast in echocardiographic and radiological ultrasound imaging.

Multiple modifications and variations of the present invention are obviously possible in light of the information above. It should therefore be understood that within the scope of the attached patent claims, the invention can be carried out differently than what is specifically described here.

Claims (18)

1. A method of preparing a phospholipid suspension, characterized in that it comprises: (a) contacting at least two lipids with a first non-aqueous solvent to form a solution; (b) concentrating the solution to a thick gel; (c) contacting the thick gel with a second non-aqueous solvent to form a solution; (d) concentrating this solution from step (c) to form a lipid mixture; (e) contacting the lipid mixture with a non-aqueous solvent, wherein the lipid mixture is substantially dissolved in the non-aqueous solvent to form a slurry; and (f) without removing the non-aqueous solvent, contacting the solution from step (e) with an aqueous solution to form a lipid suspension. 2. Method according to claim 1, characterized in that the non-aqueous solvent from step (e) is selected from propylene glycol, ethylene glycol and polyethylene glycol 300. 3. Method according to claim 2, characterized in that the lipid mixture comprises: (a) 1,2-dipalmitoyl-jrt-glycero-3-phosphatidylcholine; (b) 1,2-dipalmitoyl-sn-glycero-3-phosphotidine, monosodium salt; and (c) N-(methoxypolyethylene glycol 5000 carbamoyl)-1,2-dipalmitoyl-s'-glycero-3-phosphatidylethanolamine, monosodium salt. 4. Method according to claim 2, characterized in that the non-aqueous solvent from step (e) is heated to a temperature of approximately 30 to 70°C before it is contacted with the lipid mixture. 5. Method according to claim 2, characterized in that the ratio of lipid mixture to non-aqueous solvent from step (e) is from about 5 mg of lipid mixture per ml of non-aqueous solvent to 15 mg/ml. 6. Method according to claim 2, characterized in that the aqueous solution in step (f) is selected from water, salt solution, a salt solution/glycerin mixture, and a salt solution/glycerin/non-aqueous solvent mixture. 7. Method according to claim 6, characterized in that the aqueous solution is a salt solution, glycerin and propylene glycol mixture. 8. Method according to claim 7, characterized in that 6.8 mg/ml of sodium chloride is present, 0.1 ml/ml glycerin is present, 0.1 ml/ml propylene glycol is present, and 0.75 to 1.0 mg/ml of the lipid mixture is present. 9. Method according to claim 1, characterized in that the method further comprises: (g) heating the lipid suspension from step (f) to a temperature equal to or above the highest gel-to-liquid crystal phase transition temperature of the lipids present in the suspension. 10. Method according to claim 9, characterized in that the lipid suspension in step (g) is heated to a temperature of at least approximately 67°C. 11. Method according to claim 9, characterized in that the method further comprises: (h) filtering the lipid suspension through a sterilization filter. 12. Method according to claim 11, characterized in that the method further comprises: (i) distributing the filtered solution from step (h) into a medicine glass. 13. Method according to claim 12, characterized in that the method further comprises: (j) exchanging the gas at the top of the medicine glass from step (i) with a perfluorocarbon gas. 14. Method according to claim 13, characterized in that the perfluorocarbon gas is perfluoropropane. 15. Method according to claim 14, characterized in that the exchange of the upper gas is carried out by using a lyophilization chamber. 16. Method according to claim 13, characterized in that the method further comprises: (k) sterilizing the medicine glass from step (j). 17. Method according to claim 1, characterized in that the first non-aqueous solvent in step (a) is a mixture of methanol and toluene. 18. Method according to claim 1, characterized in that the second non-aqueous solvent in step (c) is a methyl t-butyl ether.